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Cancer-Associated Fibroblasts from Invasive Breast Cancer Have an Attenuated Capacity to Secrete Collagens

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Cancer-Associated Fibroblasts from Invasive Breast Cancer Have an Attenuated Capacity to Secrete Collagens INTERNATIONAL JOURNAL OF ONCOLOGY 45: 1479-1488, 2014 Cancer-associated fibroblasts from invasive breast cancer have an attenuated capacity to secrete collagens ZHIXUAN FU1,2*, PEIMING SONG1*, DONGBO LI3, CHENGHAO YI1, HUARONG CHEN1, SHUQIN RUAN1, ZHONG SHI1, WENHONG XU1, XIANHUA FU1 and SHU ZHENG1 1Key Laboratory of Cancer Prevention and Intervention (China National Ministry of Education), The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310009; 2Department of Surgical Oncology, Zhejiang Cancer Hospital, Hangzhou, Zhejiang 310022; 3Cardiovascular Ward of Geriatric Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, P.R. China Received April 22, 2014; Accepted June 18, 2014 DOI: 10.3892/ijo.2014.2562 Abstract. Normal fibroblasts produce extracellular stroma of invasive breast cancer. These studies showed that matrix (ECM) components that form the structural framework although CAFs from invasive breast cancer possess an activated of tissues. Cancer-associated fibroblasts (CAFs) with an activated phenotype, they secreted less collagen and induced less ECM phenotype mainly contribute to ECM deposition and construc- deposition in cancer stroma. In cancer tissue, the remodeling of tion of cancer masses. However, the stroma of breast cancer stromal structure and tumor microenvironment might, therefore, tissues has been shown to be more complicated, and the mecha- be attributed to the biological changes in CAFs including their nisms through which CAFs influence ECM deposition remain protein expression profile. elusive. In this study, we found that the activated fibroblast marker α-smooth muscle actin (α-SMA) was only present in the stroma Introduction of breast cancer tissue, and the CAFs isolated from invasive breast cancer sample remained to be activated and proliferative The main function of fibroblasts is secreting the components in passages. To further assess the difference between CAFs and of extracellular matrix (ECM). Without activation, fibroblasts normal breast fibroblasts (NFs), MALDI TOF/TOF-MS was are dormant in the ECM, however, once activated, fibroblasts used to analyze the secretory proteins of primary CAFs and could be involved in secreting higher levels of ECM compo- NFs. In total, 2,903 and 3,023 proteins were identified. Mass nents and exert a stronger proliferative effect (1,2). In the 1970s, spectrum quantitative assay and data analysis for extracellular Ryan et al originally described the activation of fibroblasts in proteins indicated that the CAFs produce less collagens and granulation tissues during wound healing progression (3,4). matrix-degrading enzymes compared with NFs. This finding Activated fibroblasts are also referred to as myofibroblasts was confirmed by western blot analysis. Furthermore, we due to their expression of α-smooth muscle actin (α-SMA), discovered that reduced collagen deposition was present in the as well as other important functions (5,6). Carcinoma tissues are mainly composed of tumor cells and stromal cells, the latter include fibroblasts, endothelial and inflammatory cells. Fibroblasts, termed cancer-associated fibro- Correspondence to: Professor Shu Zheng, Key Laboratory of blasts (CAFs) represent the most abundant cellular components Cancer Prevention and Intervention (China National Ministry of in cancer stroma. The heterotypic and multicellular interactions Education), The Second Affiliated Hospital, College of Medicine, among cells, soluble factors, signaling molecules, extracellular Zhejiang University, Hangzhou, Zhejiang 310009, P.R. China matrix construct a new biological system termed the ‘tumor E-mail: [email protected] microenvironment (TME)’. Researchers have demonstrated that tumor cells and the TME coevolve through continuous *Contributed equally paracrine communication (7), which not only creates a dynamic Abbreviations: CAFs, cancer-associated fibroblasts; NFs, normal signaling circuitry that promotes cancer initiation and progress, bread fibroblasts; ECM, extracellular matrix; MAL-DI TOF/TOF-MS, but also induces the activation of fibroblasts (8). CAFs present matrix-assisted laser desorption/ionization time of flight mass in cancer stroma exhibit an activated phenotype analogous to spectrometry; MRM, multiple reaction monitoring; CPS, counts per that of fibroblasts involved in wound healing or fibrosis (9,10). second; SMA, smooth muscle actin; PDGFR, platelet derived growth Indeed, Dvorak originally considered tumors to be ‘wounds factor receptor that do not heal’, because of the similarity with granulation tissue (11). Increasing evidence supports the role of CAFs as Key words: cancer-associated fibroblasts, extracellular matrix, tumor a key regulator of the paracrine signaling required for cancer microenvironment, mass spectrometry, desmoplasia progression (12,13). In contrast to resting fibroblasts, activated 1480 FU et al: cancer-associated fiBroBlasts secrete less collagen CAFs can be identified by their expression of vimentin,α -SMA, penicillin and 100 µg/ml streptomycin; Sigma), ascorbic acid fibroblasts activated protein (FAP), and platelet-derived growth (20 ng/ml), and FGF-basic (10 ng/ml; Sigma). After one week factor receptor (PDGFR-β) (14-16). Kalluri has indicated that of incubation in a humidified incubator with 5% CO2 atmo- up to 80% of stromal fibroblasts in breast cancer display this sphere, tissue debris was removed. Once primary cells reached activated phenotype (17). Furthermore, evidence indicates that 80% confluence, they were harvested and reseeded in a flask. CAFs neither revert back to normal fibroblasts nor undergo elimination via apoptosis (18). Characterization of CAFs/NFs. CAFs and NFs were seeded CAFs are further characterized by their production of into 24-well plates. The culture medium was removed and abundant ECM proteins, which are responsible for the stiffening cells were fixed for 20 min with SafeFix solution (Sinai) appearance of cancer (19,20). The deposition of ECM induced once 70-90% confluence was reached, followed by treating by CAFs in tumor stroma is known as desmoplasia, which has with 0.2% Triton X-100 for 15 min, and incubating in been shown to be extensive in or around tumors, particularly non-immunone goat serum for 20 min. Rabbit anti-vimentin, in human breast cancer and pancreatic carcinoma (21,22). A anti-FSP-1, anti-fibronectin antibodies and mouse anti- study has shown that the compression of ECM in turn leads to α-SMA, anti-cytokeratin (pan) antibodies (Maxin) were used the concentration of soluble factors that promote tumorigenesis in incubation at 4˚C as the primary antibody. Anti-mouse and in an autocrine and paracrine manner (13). However, Walker anti-rabbit fluorescent secondary antibodies were then applied has reported that the stroma in a range of primary breast carci- accordingly. After rinsing, cells were counterstained with nomas could vary from being predominantly cellular with little DAPI (Sigma), and images were obtained with a laser scan- collagens to being a dense collagenous stroma with apparently ning confocal microscope. Immunocytochemistry by DAB few stromal cells (23). These results are contradictory to many staining was conducted. The methods for western blot analysis research results on the universal deposition of ECM in cancer are presented in the section ‘Western blot analysis’. tissue. Therefore, it is necessary to elucidate the relationship between activated CAFs and their function in ECM deposition. Cell growth evaluation. Briefly, 1,500 CAFs and NFs were In this study, we aimed to investigate the secretomics of CAFs seeded per well in 96-well plates. Cell viability was assessed by mass spectrometry and other means in order to elucidate the daily by adding 20 µl of 3-(4,5-dimethylthiazol-2-thiazyl)- relationship between CAFs and ECM deposition in breast cancer. 2,5-diphenyl-tetrazolium bromide (MTT; 5 mg/ml in PBS) to each well. MTT was also added to the control wells without Materials and methods cells. Each sample included six replicates. Following 4-h incu- bation at 37˚C, the medium was removed. An aliquot of 200 µl Specimens. This study was based on a well-characterized DMSO was transfered to each well, and the plate was agitated series of TNM stage T2-T3 primary invasive breast carcinoma for 15 min. The absorbance at 570 nm was then detected. For cases and fibroadenomas. Invasive breast cancer was selected MTT assays that were performed on the same day with cell for study due to its typical desmoplastic response and the pres- seeding, the cells were allowed to attach for 3 h before the ence of large numbers of activated CAFs. The fresh specimens addition of MTT. Growth curves were constructed by plotting of invasive carcinoma were collected from cancer patients who absorbance (mean ± SD) against time. accepted radical mastectomy at the Second Affiliated Hospital of Zhejiang University. Written consent was obtained from all Extraction of secretory proteins from CAFs and NFs. When patients and this study was approved by the ethics committee CAFs and NFs reached 70-80% confluence, the medium was of Zhejiang University. Tissue specimens used in immunohis- removed, and the flasks were washed gently three times with tochemistry and histochemistry were obtained from the tissue PBS. Cells were then cultured in conditioned medium (CM; bank of the Cancer Institute, Zhejiang University. serum-free, phenol red-free DMEM medium supplemented with ascorbic acid 20 ng/ml). Following 18-h incubation,
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